The assay of hydrogen peroxide is relevant to the assay of biochemicals that are oxidized in enzyme catalyzed reactions by molecular oxygen where O.sub.2 is reduced to hydrogen peroxide. The hydrogen peroxide is generally assayed spectrophotometrically or electrochemically. An electrochemical assay for H.sub.2 O.sub.2 may involve electrooxidation of H.sub.2 O.sub.2, usually near +0.7V (SCE), to O.sub.2 or electroreduction, near 0.0V (SCE), to H.sub.2 O (Hall, Biosensors, Prentice Hall, Englewood Cliffs, NJ, 1991, p. 16, 135, 221, 224, 283-4; Cass, Biosensors: A Practical Approach, Oxford Univ. Press, 1990, pp. 33, 34)
Detection and quantification of a substantial number of biochemicals is also accomplished by amperometric assay relying on the selective electrooxidation of NADH and NADPH as co-factors of relevant enzymes. The electrooxidation products NAD.sup.+ or NADP.sup.+ can be enzymatically re-reduced and detected by electrocatalytic enzyme electrodes. EQU NAD(P)H .fwdarw. NAD(P).sup.+ +2e.sup.- +H.sup.+ ( 1)
The reversible potential of the NADH/NAD.sup.+ couple is - 0.55V (SCE) at pH7 (McGilvery, Biochemistry-A Functional Approach, W.B. Saunders & Co., Philadelphia, 1983, p. 404). Because this reaction involves the concerted transfer of two electrons and a proton, it is usually slow, proceeding only at high overpotentials to achieve practical rates on most electrodes. At these high overpotentials, reaction products of NAD(P)H and other constituents of biological fluids interfere with amperometric assays of NAD(P)H (Moiroux and Elving, Anal. Chem., 1979, 51:346; Blaedel and Jenkins, Anal. Chem., 1975, 47:1337).
Because of this problem, electrodes were developed on which the conversion of NAD(P)H to NAD(P).sup.+ proceeded rapidly at low overpotential (DeGrand and Miller, J. Am. Chem. Soc., 1980, 102:5728-32; Kitani et al., J. Am. Chem. Soc., 1981, 103:7636-41; Fukui et al., J. Am. Chem. Soc., 1982, 104:28; Lau and Miller, J. Am. Chem. Soc., 1983, 105:5271; Gorton et al., Anal. Chem. Acta., 1991, 250:203-48; Cenas et al., J. Electroanal. Chem. Interfacial Electrochem, 1985, 189:163; Kulys, Biosensors, 1986, 2:3). The most successful of these electrodes utilized electrode-bound, electrode-adsorbed or freely diffusing mediators having quinoid structures in their oxidized state (Gorton et al. 1991; Cenas, 1985; Kulys, 1986; Gorton et al. 1984, J. Electroanal. Chem. Interfacial Electrochem, 1984, 161:103; Persson and Gorton, J. Electroanal. Chem. Interfacial Electrochem, 1990, 292:115; Bremle et al., Electroanalysis, 1991, 3:77-86). The quinoids (Q) effectively catalyze conversion of NAD(P)H to NAD(P).sup.+ at potentials near 0.0V (SEC). EQU Q.sup.+ + NAD(P)H .fwdarw. QH + NAD.sup.+ ( 2) EQU QH + O.sub.2 + H.sup.+ .fwdarw. Q.sup.+ + H.sub.2 O.sub.2 ( 3)
In such electrodes, two electrons and a proton are transferred from NAD(P)H to a quinoid mediator (Reaction 2). A particularly effective mediator is water-soluble 5-methyl-phenazonium cation (PMS.sup.+) which is quantitatively reduced by NAD(P)H to 5-methylphenazine (PMSH). PMSH is next reoxidized to PMS.sup.+ by dissolved molecular oxygen which is, in turn, reduced to H.sub.2O.sub.2 (Reaction 3). In this reaction, each mole of NAD(P)H produces one mole of H.sub.2 O.sub.2 in the presence of dissolved molecular oxygen.
Previously reported schemes for the detection of NADH and NADPH have amperometrically sensed the L depletion of oxygen (Polster and Schmidt, Talanta, 1989, 36:864-866; Huck et al., Analyst, 1984, 109:147-150) or have spectrophotometrically measured the H.sub.2 O.sub.2 generated (Williams et al., Anal. Chem., 1976, 48:1481-84; Europ. pat. Appl. EP 317070, 1989; Europ. Pat. Appl. EP 285998, 1989) by these reactions.
Such indirect detection methods suffer problems of interference by other constituents in the test sample, and/or lack the sensitivity of a useful amperometric biosensor.
It would be highly desirable to provide an amperometric biosensor for the detection of H.sub.2 O.sub.2, NADH and NADPH which is highly sensitive and which is not significantly hampered by interfering substances in a test sample.